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PLP/DM20 ratio is regulated by hnRNPH and F and a novel G-rich enhancer in oligodendrocytes.

Wang E, Dimova N, Cambi F - Nucleic Acids Res. (2007)

Bottom Line: Knock down of hnRNPH increased PLP/DM20 ratio, while hnRNPF did not.Mutation of M2, but not ISE reduced the synergistic effect.We conclude that developmental changes in hnRNPH/F associated with OLs differentiation synergistically regulate PLP alternative splicing and a G-rich enhancer participates in the regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of Kentucky, Lexington, KY, USA.

ABSTRACT
Alternative splicing of competing 5' splice sites is regulated by enhancers and silencers in the spliced exon. We have characterized sequences and splicing factors that regulate alternative splicing of PLP and DM20, myelin proteins produced by oligodendrocytes (OLs) by selection of 5' splice sites in exon 3. We identify a G-rich enhancer (M2) of DM20 5' splice site in exon 3B and show that individual G triplets forming M2 are functionally distinct and the distal group plays a dominant role. G-rich M2 and a G-rich splicing enhancer (ISE) in intron 3 share similarities in function and protein binding. The G-rich sequences are necessary for binding of hnRNPs to both enhancers. Reduction in hnRNPH and F expression in differentiated OLs correlates temporally with increased PLP/DM20 ratio. Knock down of hnRNPH increased PLP/DM20 ratio, while hnRNPF did not. Silencing hnRNPH and F increased the PLP/DM20 ratio more than hnRNPH alone, demonstrating a novel synergistic effect. Mutation of M2, but not ISE reduced the synergistic effect. Replacement of M2 and all G runs in exon 3B abolished it almost completely. We conclude that developmental changes in hnRNPH/F associated with OLs differentiation synergistically regulate PLP alternative splicing and a G-rich enhancer participates in the regulation.

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PLP-neo construct and mutations in PLP exon 3B. (A) Schematic representation of the PLP-neo splicing construct. The arrows indicate the position of the PCR primers. The PLP and DM20 PCR products are shown. (B) Exon 3B: 349 indicates G at the invariant GT of DM20 5′ splice site, 453 is the last base of exon 3B at the PLP 5′ splice site, 409 is the position of a human mutation occurring in an ASF/SF2 motif and ESE indicates the ASF/SF2 binding motif (16). The linker scan sequence substitutions are aligned below the wild-type sequences. All mutant constructs replace 10 nt, except for M6 and M10 (see text), and are of the same length as the wild-type construct.
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Figure 1: PLP-neo construct and mutations in PLP exon 3B. (A) Schematic representation of the PLP-neo splicing construct. The arrows indicate the position of the PCR primers. The PLP and DM20 PCR products are shown. (B) Exon 3B: 349 indicates G at the invariant GT of DM20 5′ splice site, 453 is the last base of exon 3B at the PLP 5′ splice site, 409 is the position of a human mutation occurring in an ASF/SF2 motif and ESE indicates the ASF/SF2 binding motif (16). The linker scan sequence substitutions are aligned below the wild-type sequences. All mutant constructs replace 10 nt, except for M6 and M10 (see text), and are of the same length as the wild-type construct.

Mentions: Total RNA was extracted with RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and treated with the DNA-free Kit (Ambion, Austin, TX, USA). Total RNA (0.5 μg) was reverse transcribed using random hexamer primers (BD Biosciences, Palo Alto, CA, USA). The PLP and DM20 PCR products and PLP+DM20 product derived from PLP-neo were amplified and quantitated as described (16) (Figures 1A and 4A).


PLP/DM20 ratio is regulated by hnRNPH and F and a novel G-rich enhancer in oligodendrocytes.

Wang E, Dimova N, Cambi F - Nucleic Acids Res. (2007)

PLP-neo construct and mutations in PLP exon 3B. (A) Schematic representation of the PLP-neo splicing construct. The arrows indicate the position of the PCR primers. The PLP and DM20 PCR products are shown. (B) Exon 3B: 349 indicates G at the invariant GT of DM20 5′ splice site, 453 is the last base of exon 3B at the PLP 5′ splice site, 409 is the position of a human mutation occurring in an ASF/SF2 motif and ESE indicates the ASF/SF2 binding motif (16). The linker scan sequence substitutions are aligned below the wild-type sequences. All mutant constructs replace 10 nt, except for M6 and M10 (see text), and are of the same length as the wild-type construct.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919487&req=5

Figure 1: PLP-neo construct and mutations in PLP exon 3B. (A) Schematic representation of the PLP-neo splicing construct. The arrows indicate the position of the PCR primers. The PLP and DM20 PCR products are shown. (B) Exon 3B: 349 indicates G at the invariant GT of DM20 5′ splice site, 453 is the last base of exon 3B at the PLP 5′ splice site, 409 is the position of a human mutation occurring in an ASF/SF2 motif and ESE indicates the ASF/SF2 binding motif (16). The linker scan sequence substitutions are aligned below the wild-type sequences. All mutant constructs replace 10 nt, except for M6 and M10 (see text), and are of the same length as the wild-type construct.
Mentions: Total RNA was extracted with RNeasy Mini Kit (Qiagen, Valencia, CA, USA) and treated with the DNA-free Kit (Ambion, Austin, TX, USA). Total RNA (0.5 μg) was reverse transcribed using random hexamer primers (BD Biosciences, Palo Alto, CA, USA). The PLP and DM20 PCR products and PLP+DM20 product derived from PLP-neo were amplified and quantitated as described (16) (Figures 1A and 4A).

Bottom Line: Knock down of hnRNPH increased PLP/DM20 ratio, while hnRNPF did not.Mutation of M2, but not ISE reduced the synergistic effect.We conclude that developmental changes in hnRNPH/F associated with OLs differentiation synergistically regulate PLP alternative splicing and a G-rich enhancer participates in the regulation.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurology, University of Kentucky, Lexington, KY, USA.

ABSTRACT
Alternative splicing of competing 5' splice sites is regulated by enhancers and silencers in the spliced exon. We have characterized sequences and splicing factors that regulate alternative splicing of PLP and DM20, myelin proteins produced by oligodendrocytes (OLs) by selection of 5' splice sites in exon 3. We identify a G-rich enhancer (M2) of DM20 5' splice site in exon 3B and show that individual G triplets forming M2 are functionally distinct and the distal group plays a dominant role. G-rich M2 and a G-rich splicing enhancer (ISE) in intron 3 share similarities in function and protein binding. The G-rich sequences are necessary for binding of hnRNPs to both enhancers. Reduction in hnRNPH and F expression in differentiated OLs correlates temporally with increased PLP/DM20 ratio. Knock down of hnRNPH increased PLP/DM20 ratio, while hnRNPF did not. Silencing hnRNPH and F increased the PLP/DM20 ratio more than hnRNPH alone, demonstrating a novel synergistic effect. Mutation of M2, but not ISE reduced the synergistic effect. Replacement of M2 and all G runs in exon 3B abolished it almost completely. We conclude that developmental changes in hnRNPH/F associated with OLs differentiation synergistically regulate PLP alternative splicing and a G-rich enhancer participates in the regulation.

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